SiRNA molecule targeting human SLC25A3 gene and application thereof

Abstract

The invention discloses a siRNA (small interfering Ribonucleic Acid) molecule of a targeted human SLC25A3 gene, which is composed of a positive-sense strand and an antisense strand, and the specific sequence of the siRNA molecule is as follows: the positive-sense strand is 5 '-AGUACAAGGCAUAUUUAANn-3', the antisense strand is 5 '-UUAAUUAUGUACUNn-3', or the positive-sense strand is 5 '-GACUCGUGAAGGAGUCNn-3', and the antisense strand is 5 '- Wherein N in the positive-sense strand and N in the antisense strand are the same or different and are respectively and independently cytosine C, uracil U, guanine G, adenine A, deoxycytosine dC, deoxyguanine dG, deoxyadenine dA or deoxythymine dT; n represents the number of N, and n = 0, 1 or 2. The invention further discloses application of the siRNA molecule of the targeted human SLC25A3 gene, and the siRNA molecule is applied to liver cancer treatment by efficiently inhibiting the expression level of the SLC25A3.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an siRNA molecule targeting a human SLC25A3 gene, and further relates to application of the siRNA molecule in preparation of a liver cancer treatment drug. BACKGROUND

[0002] Liver cancer is one of the most common malignant tumors in the world, and its morbidity and mortality are high. At present, the clinical treatment strategies for liver cancer mainly involve surgical treatment, interventional treatment, local ablation treatment, radiotherapy and chemotherapy. Due to the insidious onset of liver cancer, early diagnosis is difficult, and most patients are found to be in the advanced stage, so the treatment effect is poor and the tumor is prone to metastasis and recurrence. As a new type of therapy, tumor molecular targeted therapy has gradually become an important means of clinical tumor treatment. Since molecular targeted therapy is to intervene (block or inhibit) key genes and signal transduction pathways of tumor occurrence through specific molecules, so as to inhibit the growth, metastasis or induce apoptosis of tumor cells, therefore, compared with traditional treatment methods, it has better precision, can selectively kill tumor cells, has less or no damage to normal tissues, has less side effects, and is not prone to drug resistance. At present, the number of molecular targeted drugs for liver cancer treatment is very limited, and the key reason is that the number of effective molecular targets is insufficient, and it is urgent to find new specific molecular targets.

[0003] The solute carrier (SLC) superfamily is the largest class of membrane transport proteins in cells. So far, 55 families have been found, containing about 362 members. They can mediate the transmembrane transport of various solutes (sugars, amino acids, nucleic acids, ions, drugs, etc.) between cells and the outside world or between cells, and thus participate in important physiological activities such as energy transmission between cells, nutrient metabolism and signal transduction. SLC25A3 (mitochondrial solute carrier family 25, member 3) is a member of the SLC25 family located in the inner membrane of mitochondria, encoding mitochondrial phosphate carrier PiC2, which is a copper and phosphate transporter. It has two isoforms, one is PiC2A produced by alternative splicing of exon 3, which is only expressed in the heart and skeletal muscle, and the other is PiC2B, which is expressed in tissues. Mitochondrial transporter SLC25A3 binds Cu ＋And transport ions across the inner membrane of mitochondria, load to mitochondrial cytochrome c oxidase (COX), help COX metalization, copper (Cu) load to mitochondrial cytochrome c oxidase (COX) is necessary for cell energy production. The content of copper (Cu) in the body is relatively stable, too low or too high is not conducive to the body, the content is too low to destroy the important metal binding enzyme function, and too high will cause abnormal cell function, cause cell death. Research found that SLC25A3 as a key mitochondrial copper transporter, it may regulate copper death in tumor, therefore, SLC25A3 is a potential target for tumor therapy. At present, whether SLC25A3 is abnormally expressed in hepatocellular carcinoma and whether it is involved in the occurrence and development of HCC is still unclear.

[0004] Small interference RNA (small interference RNA, siRNA) is a double-stranded RNA molecule with a length of about 21-25 nucleotides, which realizes precise regulation of gene expression through RNA interference (RNA interference, RNAi) mechanism. After exogenous siRNA enters the cell, it can specifically recognize and degrade the target homologous messenger RNA (mRNA), thereby inhibiting the synthesis of pathogenic protein, and then achieving the purpose of treating diseases, which is an ideal molecular targeted therapy. Studies have shown that siRNA drugs have great potential in the treatment of tumors. By June 2025, there have been 6 siRNA drugs on the market worldwide. At present, although there are several anti-tumor siRNA therapies in the clinical trial stage, there is no siRNA drug for liver cancer treatment on the market. SUMMARY

[0005] The first object of the present application is to provide an siRNA molecule targeting human SLC25A3 gene, which is applied to the treatment of liver cancer by efficiently inhibiting the expression level of SLC25A3.

[0006] The second object of the present application is to provide the use of the above-mentioned siRNA molecule in the preparation of a drug for inhibiting the expression of SLC25A3.

[0007] The third object of the present application is to provide a drug for treating liver cancer.

[0008] The fourth object of the present application is to provide a pharmaceutical preparation for treating liver cancer.

[0009] The first technical solution adopted by the present application is: an siRNA molecule targeting human SLC25A3 gene, which is composed of a sense strand and an antisense strand, and the specific sequence is as follows: Sense strand: 5'-AGUACAAGGGCAUAUUUAANn-3' Antisense strand: 5'-UUAAAUAUGCCCUUGUACUNn-3' or: Sense strand: 5'-GACUCCGUGAAGGUCUACUNn-3' Antisense strand: 5'-AGUAGACCUUCACGGAGUCNn-3' Wherein, N in the sense strand and the antisense strand is the same or different, and each independently is cytosine C, uracil U, guanine G, adenine A, deoxycytidine dC, deoxyguanosine dG, deoxyadenine dA or deoxythymidine dT; n represents the number of N, n=0, 1 or 2.

[0010] The first technical solution adopted by the present application is further characterized in that: Further, the siRNA molecule targeting the human SLC25A3 gene, n=0; The sequence of the sense strand is shown as SEQ ID NO: 1, and the sequence of the antisense strand is shown as SEQ ID NO: 2; or: The sequence of the sense strand is shown as SEQ ID NO: 3, and the sequence of the antisense strand is shown as SEQ ID NO: 4.

[0011] Further, the siRNA molecule targeting the human SLC25A3 gene, N is dT, n is 2, and the specific sequence of the siRNA molecule is as follows: Sense strand: 5'-AGUACAAGGGCAUAUUUAAdTdT-3' Antisense strand: 5'-UUAAAUAUGCCCUUGUACUdTdT-3' or: Sense strand: 5'-GACUCCGUGAAGGUCUACUdTdT-3' Antisense strand: 5'-AGUAGACCUUCACGGAGUCdTdT-3'.

[0012] The second technical solution adopted by the present application is: the application of the siRNA molecule targeting the human SLC25A3 gene in the preparation of a drug for inhibiting the expression of SLC25A3, and the drug is a drug for treating liver cancer.

[0013] The third technical solution adopted by the present application is: a drug for treating liver cancer, comprising the siRNA molecule as described above as a pharmaceutically active ingredient.

[0014] The third technical solution adopted by the present application is further characterized in that: Further, the drug is a composition, which comprises a therapeutically effective amount of the siRNA molecule as described above as an active ingredient, and further comprises a pharmaceutically acceptable carrier.

[0015] Further, the medicine is a composition, in addition to the siRNA molecule as described above, further comprising one or more other medicine components for inhibiting the expression of SLC25A3 gene.

[0016] Further, the medicine is a composition, in addition to the siRNA molecule as described above, further comprising one or more other medicine components for treating liver cancer.

[0017] Further, the medicine is an injection or a gel.

[0018] The fourth technical solution adopted by the present application is: a medicine preparation for treating liver cancer, comprising a nucleic acid sequence modifier and a carrier, wherein the nucleic acid sequence modifier is obtained by one or more of ribose modification, base modification and phosphate skeleton modification of any nucleotide of the siRNA as described above. The carrier is selected from a virus, a nanoparticle, cholesterol or a liposome.

[0019] The present application has the following beneficial effects: The siRNA molecule of the present application can significantly inhibit the expression level of SLC25A3, and can effectively inhibit the malignant progression of liver cancer cells by inhibiting the proliferation, clonogenicity, migration and invasion ability of liver cancer cells and inducing apoptosis of liver cancer cells, thereby providing a new target for the research and development of liver cancer treatment drugs. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 4 is a schematic diagram of the expression level of SLC25A3 in human normal liver cell lines and five liver cancer cell lines; Figure 2 Figure 5 is a schematic diagram of the interference efficiency of siRNA targeting SLC25A3 in liver cancer cells MHCC97L; Figure 3 Figure 6 is a schematic diagram of the effect of siRNA targeting SLC25A3 on the proliferation ability of liver cancer cells MHCC97L; Figure 4 Figure 7 is a schematic diagram of the effect of siRNA targeting SLC25A3 on the clonogenicity of liver cancer cells MHCC97L; Figure 5 Figure 8 is a schematic diagram of the effect of siRNA targeting SLC25A3 on the apoptosis of liver cancer cells MHCC97L; Figure 6a Figure 9 is a schematic diagram of the effect of siRNA targeting SLC25A3 on the migration ability of liver cancer cells MHCC97L; Figure 6b Figure 10 is a schematic diagram of the effect of siRNA targeting SLC25A3 on the invasion ability of liver cancer cells MHCC97L. DETAILED DESCRIPTION

[0021] The application will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0022] The application provides an siRNA molecule capable of efficiently inhibiting SLC25A3 gene expression, which consists of a sense strand and an antisense strand of the following sequences: Sense strand: 5'-AGUACAAGGGCAUAUUUAANn-3', Antisense strand: 5'-UUAAAUAUGCCCUUGUACUNn-3'; Or: Sense strand: 5'-GACUCCGUGAAGGUCUACUNn-3' Antisense strand: 5'-AGUAGACCUUCACGGAGUCNn-3' Wherein, N in the sense strand and the antisense strand is the same or different, and each is independently cytosine C, uracil U, guanine G, adenine A, deoxycytidine dC, deoxyguanine dG, deoxyadenine dA or deoxythymidine dT; n represents the number of N, and n is 0, 1 or 2.

[0023] The expression levels of SLC25A3 in hepatoma cell lines / normal liver cells are detected by a fluorescence quantitative PCR method, and it is found that SLC25A3 is highly expressed in hepatoma cell lines compared with normal liver cells; secondly, a plurality of siRNAs specifically targeting SLC25A3 are designed and synthesized according to the SLC25A3 gene sequence, and the hepatoma cells are transfected by a liposome-mediated method, and the efficiency of the siRNAs in inhibiting the expression of SLC25A3 is detected by a fluorescence quantitative PCR method, and at the same time, the effects of the siRNAs on cell proliferation, apoptosis, migration and invasion are detected by CCK-8, plate colony formation test, Annexin-V / PI staining flow cytometry analysis, Transwell and other experimental methods, and the results show that the two siRNAs have high silencing efficiency on SLC25A3, and can significantly inhibit the proliferation, migration and invasion ability of hepatoma cells, and promote the apoptosis of hepatoma cells, that is, significantly inhibit the progression of hepatoma cells.

[0024] The application also provides the use of the above siRNA molecules or a mixture of the two in the preparation of a medicament for inhibiting the expression of SLC25A3.

[0025] Optionally, the above medicament is an antitumor medicament.

[0026] Preferably, the above medicament is a hepatoma treatment medicament.

[0027] Wherein the siRNA molecules can be used as effective components to inhibit the proliferation, migration and invasion of hepatoma cells, or to promote the apoptosis of hepatoma cells.

[0028] The present application also provides a medicament for treating liver cancer, comprising the above-mentioned siRNA molecule as a pharmaceutically active ingredient.

[0029] Optionally, the above-mentioned medicament is a pharmaceutical composition, in addition to comprising a therapeutically effective amount of the above-mentioned siRNA molecule as an active ingredient, further comprising a pharmaceutically acceptable carrier.

[0030] The present application also provides a nucleic acid sequence modifier for use in a medicament for treating liver cancer, wherein the nucleic acid sequence modifier is obtained by one or more modifications of ribose modification, base modification and phosphate backbone modification of any nucleotide of the above-mentioned siRNA molecule.

[0031] Preferably, the above-mentioned carrier is selected from a virus, a nanoparticle, a cholesterol or a liposome.

[0032] The technical solutions of the present application will be described in further detail below in conjunction with the accompanying drawings and examples: Example 1 In one embodiment, n = 0, i.e. Sense strand: 5'-AGUACAAGGGCAUAUUUAA-3' (SEQ ID NO: 1), Antisense strand: 5'-UUAAAUAUGCCCUUGUACU-3' (SEQ ID NO: 2); Or: Sense strand: 5'-GACUCCGUGAAGGUCUACU-3' (SEQ ID NO: 3) Antisense strand: 5'-AGUAGACCUUCACGGAGUC-3' (SEQ ID NO: 4) The siRNA molecule is the backbone sequence of the group of siRNA molecules.

[0033] Example 2 In another preferred embodiment, N is dT, n = 2, i.e. Sense strand: 5'-AGUACAAGGGCAUAUUUAAdTdT-3', Antisense strand: 5'-UUAAAUAUGCCCUUGUACUdTdT-3'; Or: Sense strand: 5'-GACUCCGUGAAGGUCUACUdTdT-3' Antisense strand: 5'-AGUAGACCUUCACGGAGUCdTdT-3'.

[0034] Example 3 In one embodiment, the above-mentioned medicament is a pharmaceutical composition, which comprises a therapeutically effective amount of the above-mentioned siRNA molecule and one or more other pharmaceutical ingredients for inhibiting the expression of SLC25A3.

[0035] Example 4 In another embodiment, the above-mentioned medicament is a pharmaceutical composition, which comprises a therapeutically effective amount of the above-mentioned siRNA molecule and one or more other pharmaceutical ingredients for treating liver cancer.

[0036] Example 5 In one embodiment, the above-mentioned medicament is in the form of injection or gel. The injection is suitable for subcutaneous injection, intramuscular injection, intravenous injection or intravenous drip.

[0037] Example 6 Detection of the expression of SLC25A3 in human hepatoma cell lines and normal hepatocyte strains 1. Materials Cells: Human hepatoma cell lines Bel-7404, HepG2, Hep3B, MHCC97H, MHCC97L and normal hepatocyte strain L-O2 were purchased from the Cell Resource Center of Shanghai Life Science Research Institute, Chinese Academy of Sciences.

[0038] Reagents: DMEM medium was purchased from Hyclone, USA; fetal bovine serum was purchased from Corning, USA; RNA extraction reagent RNAiso Plus, reverse transcription reagent kit PrimeScript™ RT reagent Kit with gDNA Eraser and real-time fluorescent quantitative PCR TB Green® Premix Ex Taq™ II reagent kit were all purchased from TAKARA, Japan; qRT-PCR specific primers were synthesized by Suzhou Jinweizhi Biological Technology Co., Ltd.

[0039] 2. Methods Human hepatoma cell lines Bel-7404, HepG2, Hep3B, MHCC97H, MHCC97L and normal hepatocyte strain L-O2 were all cultured in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator at 37°C and 5% CO2.

[0040] Total RNA was extracted according to the instruction of RNAiso Plus from TAKARA. The concentration and quality of RNA were evaluated by ultramicro spectrophotometer (UPT100 from Shanghai Unico Instrument Co., Ltd.) and agarose gel electrophoresis. The following operations were carried out in two steps using the PrimeScript™ RT reagent Kit with gDNA Eraser kit from TAKARA according to the instruction: 1) removing genomic DNA in total RNA: the reaction solution (containing total RNA 1 μg, 5 × gDNA Eraser Buffer 2 μL, gDNA Eraser 1 μL, ddH2O to 10 μL) was prepared and incubated at 42℃ for 2 min in a PCR instrument and then placed on ice for use; 2) reverse transcription reaction: the reverse transcription reaction solution (containing the reaction solution of the above step 10 μL, 5 × Primescript Buffer 4 μL, Primescript RT Enzyme Mix I 1 μL, RT Primer Mix 1 μL, ddH2O 4 μL, total volume 20 μL) was further prepared based on the reaction solution obtained in the above step, and then incubated at 37℃ for 15 min in a PCR instrument and then incubated at 85℃ for 5 s to inactivate the reverse transcriptase, thereby obtaining cDNA.

[0041] TB Green Premix Ex Taq II (TliRNase H-) ®The Premix Ex Taq™ II kit was used with an ABI Q3 Real-time PCR instrument for real-time quantitative PCR detection. The qRT-PCR reaction system was as follows: 10 μL TB Green Premix Ex Taq II (2×), 1 μL forward primer (10 μM), 1 μL cDNA template, 0.4 μL ROX ReferenceDye II, 5.6 μL ddH2O, for a total volume of 20 μL. The PCR reaction conditions were as follows: 95 ℃ pre-denaturation for 30 s; 95 ℃ denaturation for 5 s, 60 ℃ annealing and extension for 30 s, for 40 cycles. Human Beta-actin gene was used as an internal control. The primers for the SLC25A3 gene are as follows: upstream primer 5'-CTGGCTCCTATGGAAGCTGCTA-3' (SEQ ID NO: 5) and downstream primer 5'-GTCTCATCCAGAGAGGAGCAAC-3' (SEQ ID NO: 6). The primer sequences for the internal reference gene Beta-actin are as follows: upstream primer 5'-TGGCACCCAGCACAATGAA-3' (SEQ ID NO: 7) and downstream primer 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3' (SEQ ID NO: 8). Using 2... -ΔΔCt The expression abundance of SLC25A3 mRNA was calculated using analytical methods.

[0042] 3. Results Figure 1 The expression levels of SLC25A3 in normal human hepatocyte cell lines and five hepatocellular carcinoma cell lines (Bel-7404, HepG2, Hep3B, MHCC97H, and MHCC97L) are shown below: P <0.01, the difference is statistically significant; The results are as follows Figure 1 As shown, compared with the normal human hepatocyte cell line L-O2, SLC25A3 was upregulated in all five human hepatocellular carcinoma cell lines, with statistically significant differences. P The result was <0.01, suggesting that the upregulation of SLC25A3 in HCC may play a pro-cancer role. The results showed that the upregulation of SLC25A3 was most significant in the MHCC97L cell line. Therefore, the MHCC97L cell line was selected as the target cell for the knockdown experiment in subsequent functional experiments.

[0043] Example 7 Effects of siRNA inhibition of SLC25A3 expression on liver cancer cell function 1. Materials Cell: Human hepatocellular carcinoma cell line MHCC97L was purchased from the Cell Resource Center of Shanghai Life Science Research Institute, Chinese Academy of Sciences.

[0044] Reagents: Transfection reagent Lipofectamine 3000 was purchased from Thermo Fisher Scientific, CCK-8 kit was purchased from Dojindo, Japan; Transwell chamber (for 24-well plate, PET membrane, 8 μm) was purchased from CORNING, USA; Crystal violet was purchased from Beijing Solabio Technology Co., Ltd.; Apoptosis kit was purchased from Dojindo, Japan.

[0045] 2、Method 2.1 Design and synthesis of siRNA sequence targeting SLC25A3 Two different splice variants of SLC25A3 cDNA sequences (NM_005888.4 and NM_213611.3) were obtained from the NCBI database. BLOCK-iT RNAi Designer software (Thermo Fisher Scientific) was used to design specific siRNA targeting SLC25A3 (siSLC25A3) based on the common exon region of different splice variants. A total of 3 siRNAs were selected from the results for synthesis. The specific siRNA sequences were composed of the following sense and antisense sequences: siRNA-1 sense strand: 5'-AGUACAAGGGCAUAUUUAA-3' (SEQ ID NO: 1), siRNA-1 antisense strand: 5'-UUAAAUAUGCCCUUGUACU-3' (SEQ ID NO: 2); or siRNA-2 sense strand: 5'-CUCUGGCGCACAUCACUAU-3' (SEQ ID NO: 9) siRNA-2 antisense strand: 5'-AUAGUGAUGUGCGCCAGAG-3' (SEQ ID NO: 10); or siRNA-3 sense strand: 5'-GACUCCGUGAAGGUCUACU-3' (SEQ ID NO: 3) siRNA-3 antisense strand: 5'-AGUAGACCUUCACGGAGUC-3' (SEQ ID NO: 4) The above siRNA targeting SLC25A3 was synthesized by Guangzhou Ribobio Biotech Co., Ltd.

[0046] The negative control sequence (siNC, no active target site on the human genome) used in the experiment was purchased from Guangzhou Ribobio Biotech Co., Ltd.

[0047] 2.3 Cell transfection Human hepatocellular carcinoma cell line MHCC97L was inoculated in 6-well plates and cultured at 37°C, 5% CO2 overnight to make the cell confluence reach about 30% before transfection. Transfection was performed according to the instructions of Lipofectamine 3000 (Thermo Fisher Scientific). The specific steps are as follows: 1) 5 μL siSLC25A3 / siNC (final concentration 50 nm) and 3.75 μL Lipofectamine 3000 transfection reagent were added to 125 μL serum-free DMEM culture solution respectively, and after mixing respectively, the siRNA-containing DMEM culture solution was added to the DMEM culture solution containing Lipofectamine 3000 transfection reagent, and carefully mixed, and then stood for 5 min to obtain the transfection solution; 2) Transfection: the above transfection solution was added to the above 6-well plate (containing 2 mL culture solution) and incubated in a 37°C, 5% CO2 incubator; 3) After 24 h of transfection, the cells were collected, and qRT-PCR was used to detect the interference effect of siRNA on SLC25A3, or cell proliferation, clone formation, apoptosis, migration and invasion experiments were performed.

[0048] 2.4 qRT-PCR detection of the interference effect of siRNA on SLC25A3 The transfected experimental group cells (liver cancer cells transfected with siSLC25A3) and control group cells (liver cancer cells transfected with siNC) were collected, and total RNA extraction, reverse transcription and qRT-PCR were performed, following the method of Example 6.

[0049] 2.5 CCK-8 cell proliferation activity detection experiment The experimental group and control group cells transfected with siSLC25A3 and siNC (50 nm) for 24 h were collected, resuspended with complete culture solution, counted, and inoculated in a 96-well plate at a density of 3000 cells / well. 100 μL / well of PBS was added to the peripheral wells, and the plate was incubated in a 37°C, 5% CO2 incubator. At the set time points (0 h, 24 h, 48 h, 72 h, 96 h), 10 μL of CCK-8 reagent was added to the test wells, and the plate was further incubated for 1 h 30 min. Then, the absorbance value of each well was measured using a multifunctional enzyme label instrument at a wavelength of 450 nm. Complete culture medium was added to the wells without cells as zero adjustment wells. 2.6 Clone formation test The experimental group and control group cells were collected 24 h after transfection of siSLC25A3 and siNC (50 nm), resuspended with complete culture medium, counted, and inoculated in a 6-well plate at a density of 500 cells per well, 3 replicates per group, and cultured in a 37°C, 5% CO2 incubator for 7 days until clear cell colonies were visible to the naked eye. The culture medium in each well was removed, washed with PBS twice, fixed with 1 mL of 4% paraformaldehyde per well for 25 min, the paraformaldehyde was removed, washed with PBS twice, and 1 mL of 0.1% crystal violet was added per well for staining for 10 min, then removed and washed with tap water for 6 min. After drying, the plate was photographed and the colonies were counted.

[0050] 2.7 Apoptosis experiment The cells were inoculated in a 6-well plate, and the experimental group and control group cells were collected 48 h after transfection of siSLC25A3 and siNC (50 nm). After washing and centrifugation with pre-cooled PBS, 195 μL of Annexin V-FITC binding solution was added to resuspend the cells, followed by the addition of 5 μL of Annexin V-FITC and 10 μL of propidium iodide (PI). After incubation at room temperature for 15 min in the dark, the apoptosis results were detected by flow cytometry within 1 h.

[0051] 2.8 Transwell experiment The experimental group and control group cells were collected 24 h after transfection of siSLC25A3 and siNC (50 nm), resuspended with complete culture medium, counted, and 200 μl of cell suspension (containing about 1×10 4 ) was inoculated in the upper chamber of a Transwell chamber with an 8 μm pore size without or with pre-coated Matrigel gel. Another 24-well plate was prepared by adding 500 μl of DMEM medium containing 20% serum to form the lower chamber. Then the Transwell chamber was placed in the 24-well plate and cultured in a 37°C, 5% CO2 incubator for 24-48 h. After the culture ended, the chamber was removed, washed with PBS 3 times, and then the cells were fixed with methanol. After air-drying, the chamber was stained with 600 μL of 0.1% crystal violet for 10 min, then washed with PBS 3 times, and the chamber was inverted and dried. Using an inverted microscope, 5 random fields were selected for observation and photography in each chamber, and the number of cells that passed through the microporous membrane was counted.

[0052] 3. Results 3.1 Interference efficiency of siSLC25A3 on SLC25A3 in liver cancer cells Figure 2 The interference efficiency of siRNA targeting SLC25A3 in liver cancer cells MHCC97L was determined. P<0.01, the difference was statistically significant; As Figure 2 shown, compared with the control group transfected with siNC, the expression of SLC25A3 in the liver cancer cells transfected with the three groups of siRNA molecules specifically targeting SLC25A3 was significantly inhibited, among which the inhibition efficiency of siRNA-1 and siRNA-3 on SLC25A3 was the most significant, and the inhibition efficiency could reach 72% and 64%, respectively. The inhibition efficiency of siRNA-2 on SLC25A3 was relatively the lowest, and the knockdown efficiency also reached 52%. Therefore, siRNA-1 and siRNA-3 were selected for subsequent inhibition of SLC25A3 expression and detection of the effect of SLC25A3 knockdown on the proliferation, apoptosis, migration and invasion ability of liver cancer cells.

[0053] 3.2 Effect of siSLC25A3 on the proliferation and clonogenicity of liver cancer cells Figure 3 Effect of siRNA targeting SLC25A3 on the proliferation of liver cancer cells MHCC97L, wherein: P <0.05, P <0.01, the difference was statistically significant; As Figure 3 shown, compared with the siNC control group, the proliferation activity of liver cancer cells in the siRNA-1 and siRNA-3 transfected groups was significantly inhibited ( P all <0.05, the difference was statistically significant).

[0054] Figure 4 Effect of siRNA targeting SLC25A3 on the clonogenicity of liver cancer cells MHCC97L, wherein: P <0.01, the difference was statistically significant; As Figure 4 shown, the results of the plate colony formation test were consistent with those of the CCK-8 test, and the plate colony formation ability of liver cancer cells in the siRNA-1 and siRNA-3 transfected groups was significantly weakened ( P all <0.01, the difference was statistically significant). In summary, transfection of siRNA targeting SLC25A3 can significantly inhibit the proliferation of liver cancer cells.

[0055] 3.3 Effect of siSLC25A3 on the apoptosis of liver cancer cells Figure 5 Effect of siRNA targeting SLC25A3 on the apoptosis of liver cancer cells MHCC97L, wherein: P <0.05, P <0.01, the difference was statistically significant; AsFigure 5 As shown, compared with the control group transfected with siNC, the apoptosis rate of liver cancer cells in the siRNA-1 and siRNA-3 transfected groups was significantly increased. P (All values ​​<0.05, indicating statistical significance) This suggests that transfection with siRNA targeting SLC25A3 can significantly induce apoptosis in liver cancer cells.

[0056] 3.4 Effects of siSLC25A3 on the migration and invasion abilities of liver cancer cells like Figure 6a and Figure 6b As shown, compared with the control group transfected with siNC, the number of liver cancer cells that migrated and invaded was significantly reduced in the groups transfected with siRNA-1 and siRNA-3. P (All values ​​<0.05, indicating statistical significance) This suggests that transfection with siRNA targeting SLC25A3 can significantly inhibit the migration and invasion of liver cancer cells.

[0057] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

[0058] sequence list sequence list <110> Xi'an Peihua University <120> siRNA molecules targeting the human SLC25A3 gene and their applications <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> RNA <213> Artificial sequence <400> 1 aguacaaggg cauauuuaa 19 <210> 2 <211> 19 <212> RNA <213> Artificial sequence <400>2 uuaaauaugc ccuuguacu 19 <210>3 <211>19 <212> RNA <213> Artificial Sequence <400>3 gacuccguga aggucuacu 19 <210>4 <211>19 <212> RNA <213> Artificial Sequence <400>4 aguagaccuu cacggaguc 19 <210>5 <211>22 <212> DNA <213> Artificial Sequence <400>5 ctggctccta tggaagctgc ta 22 <210>6 <211>22 <212> DNA <213> Artificial Sequence <400>6 gtctcatcca gagaggagca ac 22 <210>7 <211>19 <212> DNA <213> Artificial Sequence <400>7 tggcacccag cacaatgaa 19 <210>8 <211>25 <212> DNA <213> Artificial Sequence <400>8 ctaagtcata gtccgcctag aagca 25 <210>9 <211>19 <212> RNA <213> Artificial Sequence <400>9 cucuggcgca caucacuau 19 <210>10 <211>19 <212>RNA <213>Artificial Sequence <400>10 auagugaugu gcgccagag 19

Claims

1. A siRNA molecule targeting the human SLC25A3 gene, characterized in that, It consists of a justice chain and an antithesis chain, with the specific sequence as follows: Chain of Justice: 5'-AGUACAAGGGCAUAUUUAANn-3' Antonym chain: 5'-UUAAAUAUGCCCUUGUACUNn-3' or: Chain of Justice: 5'-GACUCCGUGAAGGUCUACUNn-3' Antonym chain: 5'-AGUAGACCUUCACGGAGUCNn-3' In this chain, N in the sense chain and the antisense chain may be the same or different, and each is independently cytosine C, uracil U, guanine G, adenine A, deoxycytosine dC, deoxyguanine dG, deoxyadenine dA, or deoxythymidine dT; n represents the number of N, n=0, 1, or 2.

2. The siRNA molecule targeting the human SLC25A3 gene according to claim 1, characterized in that, Where n=0; The sense chain sequence is shown in SEQ ID NO: 1, and the antisense chain sequence is shown in SEQ ID NO: 2; or: The sense chain sequence is shown in SEQ ID NO: 3, and the antisense chain sequence is shown in SEQ ID NO:

4.

3. The siRNA molecule targeting the human SLC25A3 gene according to claim 1, characterized in that, Where N is dT, n is 2, and the specific sequence of the siRNA molecule is as follows: Chain of Justice: 5'-AGUACAAGGGCAUAUUUAAdTdT-3' Antisense chain: 5'-UUAAAUAUGCCCUUGUACUdTdT-3' or: Chain of Justice: 5'-GACUCCGUGAAGGUCUACUdTdT-3' Antonym chain: 5'-AGUAGACCUUCACGGAGUCdTdT-3'.

4. The use of the siRNA molecule targeting the human SLC25A3 gene according to any one of claims 1-3 in the preparation of a drug for inhibiting SLC25A3 expression, said drug being a treatment for liver cancer.

5. A drug for treating liver cancer, characterized in that, Includes as claimed in claim 1 The siRNA molecule described in any one of the three options is used as the active pharmaceutical ingredient.

6. The drug according to claim 5, characterized in that, The drug is a composition comprising a therapeutically effective amount as claimed in claim 1. In addition to the siRNA molecule described in any one of the 3, which is an active ingredient, there is also a pharmaceutically acceptable carrier.

7. The drug according to claim 5, characterized in that, The drug is a composition comprising a therapeutically effective amount as claimed in claim 1. In addition to the siRNA molecule described in any one of the three, it also includes one or more other drug components that inhibit the expression of the SLC25A3 gene.

8. The drug according to claim 5, characterized in that, The drug is a composition comprising a therapeutically effective amount as claimed in claim 1. In addition to the siRNA molecule described in any one of the three, it also includes one or more other drug components for treating liver cancer.

9. The drug according to claim 5, characterized in that, The drug dosage form is an injection or a gel.

10. A pharmaceutical preparation for treating liver cancer, characterized in that, It includes a nucleic acid sequence modifier and a vector, wherein the nucleic acid sequence modifier is as described in claim 1. The siRNA described in any one of the 3 is obtained by performing one or more modifications of any nucleotide ribose modification, base modification, and phosphate backbone modification; The carrier is selected from viruses, nanoparticles, cholesterol, or liposomes.

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